Interactions between Shh pathway regulators and fetal alcohol exposure in mice

描述（由申请人提供）：前脑无裂畸形（HPE）是一种常见的前脑和中脸先天缺陷，有环境和遗传原因。环境因素之一是孕妇在怀孕早期饮酒。此外，在人类HPE中发现了Sonic hedgehog （Shh）信号通路的突变。突变携带者的HPE表型是高度可变的，其影响范围从前脑和中脸的严重缺陷到无临床表现。造成这种差异的原因尚不清楚，但胎儿发育过程中可能还需要其他因素，包括遗传影响或环境暴露。基因与环境的相互作用很可能与HPE这样的复杂疾病有关；然而，很少有这样的模型。Cdo和Boc是结合Shh的细胞表面蛋白，在胚胎发育的特定区域调节通路信号强度。Cdo突变小鼠的HPE表现出菌株特异性的严重程度：129/Sv背景突变小鼠的HPE表现出非常轻微的低频率HPE，而C57BL/6背景突变小鼠的HPE表现出更严重的高频率HPE。Boc突变小鼠无明显影响，但129/Sv Cdo；Boc双突变体表现出严重的颅面HPE特征。我们报道129/Sv小鼠对乙醇诱导的HPE具有抗性；然而，80%的129/Sv Cdo突变胚胎在子宫内暴露于乙醇中，显示出强烈的颅面HPE，与Cdo惊人的相似；Boc双突变体。此外，在乙醇处理的Cdo突变胚胎的前脑中，Shh的表达明显减少。因此，Cdo的损失和胎儿乙醇暴露协同产生HPE；这个事实，再加上乙醇处理过的Cdo突变胚胎与Cdo非常相似；Boc双突变体，认为Cdo的损失和乙醇暴露对Shh信号传导有协同作用。肢体和手指缺陷也与人类胎儿酒精暴露有关，C57BL/6胚胎在子宫酒精暴露会导致四肢和手指后侧畸形。所有四肢和手指的后侧图案都需要Shh。在基因敏感的背景下，失去Boc而不是Cdo会导致严重的手指缺陷。据推测，与129/Sv Cdo突变小鼠和HPE相似，129/Sv Boc突变小鼠也会对乙醇诱导的肢体/手指异常敏感。关于乙醇如何破坏肢体/手指模式的已知调节因子的信息相对较少，乙醇处理的C57BL/6和129/Sv小鼠的发育遗传分析将揭示这一过程。本研究的目的是：1)分析乙醇诱导的129/Sv Cdo突变小鼠的HPE；2)探讨乙醇诱导的129/Sv Cdo突变小鼠前脑Shh表达下调的机制；3)分析Shh通路在乙醇诱导的肢体/手指图案缺陷中的作用。这些研究包括分析一种新开发的模型，即在一种常见的出生缺陷中，一个确定的发育突变和乙醇之间的基因-环境相互作用。这些信息可能对公共卫生产生重要影响，并最终有助于对突变携带者进行咨询。

DESCRIPTION (provided by applicant): Holoprosencephaly (HPE) is a common birth defect of the forebrain and midface with both environmental and genetic causes. Among the environmental factors is maternal alcohol consumption during early pregnancy. In addition, mutations in the Sonic hedgehog (Shh) signaling pathway have been identified in human HPE. The HPE phenotype of mutation carriers is highly variable, with the spectrum of effects ranging in a continuum from severe defects in the forebrain and midface to no clinical manifestation. The reason for this variability is un- known, but factors that may also be required include additional genetic influences or environmental exposures, during fetal development. Gene-environment interactions are likely to be involved in a complex malady like HPE; however, there are few models for this. Cdo and Boc are cell surface proteins that bind Shh and regulate pathway signaling strength in specific regions of the developing embryo. Cdo mutant mice display HPE with strain-specific severity: mutants on the 129/Sv background show very mild HPE with low frequency, while mutants on the C57BL/6 background show more severe forms at high frequency. Boc mutant mice are without overt effect, but 129/Sv Cdo;Boc double-mutants display severe craniofacial HPE features. We report that 129/Sv mice are resistant to ethanol-induced HPE; however, 80% of 129/Sv Cdo mutant embryos exposed in utero to ethanol display strong craniofacial HPE and bear a striking resemblance to Cdo;Boc double mutants. Furthermore, there is a strong reduction of Shh expression in the forebrain of ethanol-treated Cdo mutant embryos. Loss of Cdo and fetal ethanol exposure therefore synergize to produce HPE; this fact, plus that ethanol-treated Cdo mutant embryos are so similar to Cdo;Boc double-mutants, argues that loss of Cdo and ethanol exposure have a synergistic effect on Shh signaling. Limb and digit defects are also associated with human fetal alcohol exposure, and in utero ethanol exposure of C57BL/6 embryos results in malformations of the posterior aspects of limbs and digits. Shh is required for all posterior patterning of limbs and digits. Loss of Boc, but not Cdo, results in severe digit defects on a genetically-sensitized background. It is hypothesized that, similar to 129/Sv Cdo mutant mice and HPE, 129/Sv Boc mutant mice will be sensitized to ethanol-induced limb/digit anomalies. There is relatively little information available on how ethanol disrupts the known regulators of limb/digit patterning, and developmental genetic analyses of ethanol-treated C57BL/6 and 129/Sv mice will shed light on this process. The aims of this proposal are: 1) to analyze ethanol-induced HPE in 129/Sv Cdo mutant mice; 2) to identify mechanisms of ethanol-induced down-regulation of Shh expression in the forebrains of 129/Sv Cdo mutant mice; and 3) to analyze the role of the Shh pathway in ethanol-induced defects in limb/digit patterning. These studies involve analysis of a newly developed model of gene-environment interaction between a defined developmental mutation and ethanol in a common birth defect. Such information may have important public health impact and could ultimately aid in the counseling of mutation carriers. PUBLIC HEALTH RELEVANCE: Maternal alcohol consumption during early pregnancy can result in birth defects, and this may be influenced by genetic variation in the human population. This application focuses on a mouse model in which a combination of in utero alcohol exposure and a specific mutation leads to a devastating and common birth defect of the forebrain and midface, called holoprosencephaly. Studies on this model, and its extension to additional ethanol-associated birth defects, are proposed, with the goal of shedding light on how genetic susceptibility may influence the outcome of fetal alcohol exposure.